Interleukin-1 beta converting enzyme like apoptosis protease-3 and 4

ABSTRACT

Disclosed are human interleukin-1 β converting enzyme like apoptosis proteases-3 and 4 and DNA (RNA) encoding such polypeptides. Also provided is a procedure for producing such polypeptides by recombinant techniques and antibodies and antagonists against such polypeptides. Also provided are methods of using the polypeptides, for example, as an antitumor agent, and antiviral agent, and antibodies and antagonists against such polypeptides for example, for treating Alzheimer&#39;s disease, Parkinson&#39;s disease, rheumatoid arthritis and head injury.

[0001] This invention relates to newly identified polynucleotides,polypeptides encoded by such polynucleotides, the use of suchpolynucleotides and polypeptides, as well as the production of suchpolynucleotides and polypeptides. More particularly, the polypeptides ofthe present invention are interleukin-1β converting enzyme likeapoptosis protease-3 and interleukin-1β converting enzyme like apoptosisprotease-4, sometimes hereinafter referred to collectively as “ICE-LAP-3and 4”. The invention also relates to inhibiting the action of suchpolypeptides.

[0002] It has recently been discovered that an interleukin-1 βconverting enzyme (ICE) is responsible for cleaving pro-IL-1 β intomature and active IL-1 β and is also responsible for programmed celldeath (or apoptosis), which is a process through which organisms get ridof unwanted cells. The present invention is directed to ICE-LAP-3 and 4which are structurally related to ICE.

[0003] In the nematode caenorhabditis elegans, a genetic pathway ofprogrammed cell death has been identified (Ellis, R. E., et al. Annu.Rev. Cell Biol., 7:663-698 (1991)). Two genes, ced-3 and ced-4, areessential for cells to undergo programmed cell death in C. elegans(Ellis, H. M., and Horvitz, H. R., Cell, 44:817-829 (1986)). Recessivemutations that eliminate the function of these two genes prevent normalprogrammed cell death during the development of C. elegans. The knownvertebrate counterpart to ced-3 protein is ICE. The overall amino acididentity between ced-3 and ICE is 28%, with a region of 115 amino acids(residues 246-360 of ced-3 and 164-278 of ICE) that shows the highestidentity (43%). This region contains a conserved pentapeptide, QACRG(residues 356-360 of ced-3), which contains a cysteine known to beessential for ICE function. The ICE-LAP-3 and 4 polypeptides of thepresent invention also have the same conserved pentapeptide and thecysteine residue which is essential for ICE function.

[0004] The similarity between ced-3 and ICE suggests not only that ced-3might function as a cysteine protease but also that ICE might act as avertebrate programmed cell death gene. ced-3 and the vertebratecounterpart, ICE, control programmed cell death during embryonicdevelopment, (Gagliarnini, V. et al., Science, 263:826:828 (1994).

[0005] ICE mRNA has been detected in a variety of tissues, includingperipheral blood monocytes, peripheral blood lymphocytes, peripheralblood neutrophils, resting and activated peripheral blood T lymphocytes,placenta, the B lymphoblastoid line CB23, and monocytic leukemia cellline THP-1 cells (Cerretti, D. P., et al., Science, 256:97-100 (1992)),suggesting that ICE may have an additional substrate in addition topro-IL-1β. The substrate that ICE acts upon to cause cell death ispresently unknown. One possibility is that it may be a vertebratehomolog of the C. elegans cell death gene ced-4. Alternatively, ICEmight directly cause cell death by proteolytically cleaving proteinsthat are essential for cell viability.

[0006] The mammalian gene bc1-2, has been found to protect immune cellscalled lymphocytes from cell suicide. Also, crmA, a cow pox virus geneprotein product inhibits ICE's protein splitting activity.

[0007] The polypeptides of the present invention have been putativelyidentified as ICE-LAP-3 and 4. This identification has been made as aresult of amino acid sequence homology.

[0008] In accordance with one aspect of the present invention, there areprovided novel mature polypeptides which are ICE-LAP-3 and 4, as well asfragments, analogs and derivatives thereof. The polypeptides of thepresent invention are of human origin.

[0009] In accordance with another aspect of the present invention, thereare provided polynucleotides (DNA or RNA) which encode suchpolypeptides.

[0010] In accordance with yet a further aspect of the present invention,there is provided a process for producing such polypeptides byrecombinant techniques.

[0011] In accordance with yet a further aspect of the present invention,there is provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides for therapeutic purposes, forexample, as an antiviral agent, an anti-tumor agent and to controlembryonic development and tissue homeostasis.

[0012] In accordance with yet a further aspect of the present invention,there is provided an antibody against such polypeptides.

[0013] In accordance with yet another aspect of the present invention,there are provided antagonists to such polypeptides, which may be usedto inhibit the action of such polypeptides, for example, in thetreatment of Alzheimer's disease, Parkinson's disease, rheumatoidarthritis, septic shock and head injuries.

[0014] These and other aspects of the present invention should beapparent to those skilled in the art from the teachings herein.

[0015] The following drawings are illustrative of embodiments of theinvention and are not meant to limit the scope of the invention asencompassed by the claims.

[0016]FIG. 1 shows the cDNA and corresponding deduced amino acidsequence of ICE-LAP-3. The polypeptide encoded by the amino acidsequence shown is the putative mature form of the polypeptide (minus theinitial methionine residue), and the standard one-letter abbreviationfor amino acids is used.

[0017]FIG. 2 shows the cDNA and corresponding deduced amino acidsequence of ICE-LAP-4. The polypeptide encoded by the amino acidsequence shown is the putative mature form of the polypeptide (minus theinitial methionine residue).

[0018]FIG. 3 shows an amino acid sequence comparison between ICE-LAP-3,ICE-LAP-4, human ICE and the C. elegan cell death gene ced-3. Shadedareas represent amino acid matches between the different sequences.

[0019] Sequencing inaccuracies are a common problem when attempting todetermine polynucleotide sequences. Accordingly, the sequences of FIGS.1 and 2 are based on several sequencing runs and the sequencing accuracyis considered to be at least 97%.

[0020] In accordance with an aspect of the present invention, there areprovided isolated nucleic acids (polynucleotides) which encode themature polypeptides having the deduced amino acid sequence of FIGS. 1and 2 or for the mature polypeptide encoded by the cDNA of the clonesdeposited as ATCC Deposit No. 75875 encoding ICE-LAP-3, and ATCC DepositNo. 75873 encoding ICE-LAP-4, which were deposited Aug. 25, 1994.

[0021] The polynucleotide encoding ICE-LAP-3 can be detected from humanprostate, human endometrial tumor, human pancreatic tumor, human adrenalgland tumor and human tonsil. The full-length encoding ICE-LAP-3 wasdiscovered in a cDNA library derived from human endometrial tumor. It isstructurally related to the Interleukin-1β converting enzyme family. Itcontains an open reading frame encoding a protein of approximately 341amino acid residues. The protein exhibits the highest degree of homologyto C. elegans cell death gene ced-3 which is a hoholog of humaninterleukin-1 β converting enzyme, with 68% similarity and 43% identityover the entire amino acid sequence. It should be pointed out that thepentapeptide QACRG is conserved and is located at amino acid position259-263.

[0022] The polynucleotide encoding ICE-LAP-4 was discovered in a cDNAlibrary derived from human tonsils. It is structurally related to theICE family. It contains an open reading frame encoding a protein ofabout 277 amino acid residues. The protein exhibits the highest degreeof homology to the C. elegans cell death gene ced-3 with 29% identityand 46% similarity over a 277 amino acid stretch. It is also importantthat the pentapeptide QACRG is conserved and is located at aminoposition 161-165.

[0023] The polynucleotides of the present invention may be in the formof RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, andsynthetic DNA. The DNA may be double-stranded or single-stranded, and ifsingle stranded may be the coding strand or non-coding (anti-sense)strand. The coding sequence which encode the mature polypeptides may beidentical to the coding sequence shown in FIGS. 1 and 2 or that of thedeposited clones or may be a different coding sequence which codingsequence, as a result of the redundancy or degeneracy of the geneticcode, encode the same mature polypeptides, and derivatives thereof, asthe DNA of FIGS. 1 and 2 or the deposited cDNA.

[0024] The polynucleotides which encode for the mature polypeptides ofFIGS. 1 and 2 or for the mature polypeptides encoded by the depositedcDNAs may include: only the coding sequence for the mature polypeptide;the coding sequence for the mature polypeptide (and optionallyadditional coding sequence) and non-coding sequence, such as introns ornon-coding sequence 5′ and/or 3′ of the coding sequence for the maturepolypeptide.

[0025] Thus, the term “polynucleotide encoding a polypeptide”encompasses a polynucleotide which includes only coding sequence for thepolypeptide as well as a polynucleotide which includes additional codingand/or non-coding sequence.

[0026] The present invention further relates to variants of thehereinabove described polynucleotides which encode for fragments,analogs and derivatives of the polypeptides having the deduced aminoacid sequence of FIGS. 1 and 2 or the polypeptides encoded by the cDNAof the deposited clones. The variants of the polynucleotides may benaturally occurring allelic variants of the polynucleotides ornon-naturally occurring variants of the polynucleotides.

[0027] Thus, the present invention includes polynucleotides encoding thesame mature polypeptides as shown in FIGS. 1 and 2 or the same maturepolypeptides encoded by the cDNA of the deposited clones as well asvariants of such polynucleotides which variants encode for a fragment,derivative or analog of the polypeptides of FIGS. 1 and 2 or thepolypeptides encoded by the cDNA of the deposited clones. Suchnucleotide variants include deletion variants, substitution variants andaddition or insertion variants.

[0028] As hereinabove indicated, the polynucleotides may have a codingsequence which is a naturally occurring allelic variant of the codingsequence shown in FIGS. 1 and 2 or of the coding sequence of thedeposited clones. As known in the art, an allelic variant is analternate form of a polynucleotide sequence which may have asubstitution, deletion or addition of nucleotides, which does notsubstantially alter the function of the encoded polypeptides.

[0029] The polynucleotides of the present invention may also have thecoding sequence fused in frame to a sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptides fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

[0030] The present invention further relates to polynucleotides whichhybridize to the hereinabove-described sequences if there is at least50% and preferably 70% identity between the sequences. The presentinvention particularly relates to polynucleotides which hybridize understringent conditions to the hereinabove-described polynucleotides. Asherein used, the term “stringent conditions” means hybridization willoccur only if there is at least 95% and preferably at least 97% identitybetween the sequences. The polynucleotides which hybridize to thehereinabove described polynucleotides in a preferred embodiment encodepolypeptides which retain substantially the same biological function oractivity as the mature polypeptides encoded by the cDNA of FIGS. 1 and 2or the deposited cDNAs.

[0031] The deposit(s) referred to herein will be maintained under theterms of the Budapest Treaty on the International Recognition of theDeposit of Micro-organisms for purposes of Patent Procedure. Thesedeposits are provided merely as convenience to those of skill in the artand are not an admission that a deposit is required under 35 U.S.C.§112. The sequence of the polynucleotides contained in the depositedmaterials, as well as the amino acid sequence of the polypeptidesencoded thereby, are incorporated herein by reference and arecontrolling in the event of any conflict with any description ofsequences herein. A license may be required to make, use or sell thedeposited materials, and no such license is hereby granted.

[0032] The present invention further relates to ICE-LAP-3 and 4polypeptides which have the deduced amino acid sequence of FIGS. 1 and 2or which has the amino acid sequence encoded by the deposited cDNAs, aswell as fragments, analogs and derivatives of such polypeptides.

[0033] The terms “fragment,” “derivative” and “analog” when referring tothe polypeptides of FIGS. 1 and 2 or that encoded by the deposited cDNA,means polypeptides which retain essentially the same biological functionor activity as such polypeptides, and wherein derivatives includepolypeptides with enhanced or reduced biological function. An analogincludes a proprotein which can be activated by cleavage of theproprotein portion to produce active mature polypeptides.

[0034] The polypeptides of the present invention may be recombinantpolypeptides, natural polypeptides or synthetic polypeptides, preferablyrecombinant polypeptides.

[0035] The fragment, derivative or analog of the polypeptides of FIGS. 1and 2 or that encoded by the deposited cDNAs may be (i) one in which oneor more of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol). Such fragments, derivatives and analogs are deemedto be within the scope of those skilled in the art from the teachingsherein.

[0036] The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity. The term “isolated” means that the material is removed fromits original environment (e.g., the natural environment if it isnaturally occurring). For example, a naturally-occurring polynucleotideor polypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

[0037] The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

[0038] Host cells are genetically engineered (transduced or transformedor transfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the ICE-LAP-3 and 4 genes. The cultureconditions, such as temperature, pH and the like, are those previouslyused with the host cell selected for expression, and will be apparent tothe ordinarily skilled artisan.

[0039] The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

[0040] The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

[0041] The DNA sequence in the expression vector is operatively linkedto an appropriate expression control sequence(s) (promoter) to directmRNA synthesis. As representative examples of such promoters, there maybe mentioned: LTRs from retroviruses, e.g. RSV, HIV, HTLVI, CMV or SV40promoter, the E. coli. lac or trp, the phage lambda P_(L) promoter andother promoters known to control expression of genes in prokaryotic oreukaryotic cells or their viruses. However, also cellular signals can beused, for example, human-β-actin-promoter). The expression vector cancontain a ribosome binding site for translation initiation and atranscription terminator. The vector may also include appropriatesequences for amplifying the copy number of the gene.

[0042] In addition, the expression vectors preferably contain one ormore selectable marker genes to provide a phenotypic trait for selectionof transformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

[0043] The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

[0044] As representative examples of appropriate hosts, there may bementioned: bacterial cells, such as E. coli, Bacillus subtilis,Streptomyces, Salmonella typhimurium; fungal cells, such as yeast;insect cells such as Drosophila and Sf9; animal cells such as CHO, COS,HEK 293 or Bowes melanoma; plant cells, etc. The selection of anappropriate host is deemed to be within the scope of those skilled inthe art from the teachings herein.

[0045] More particularly, the present invention also includesrecombinant constructs comprising one or more of the sequences asbroadly described above. The constructs comprise a vector, such as aplasmid or viral vector, into which a sequence of the invention has beeninserted, in a forward or reverse orientation. In a preferred aspect ofthis embodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

[0046] Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

[0047] In a further embodiment, the present invention relates to hostcells containing the above-described constructs. The host cell can be ahigher eukaryotic cell, such as a mammalian cell, or a lower eukaryoticcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell. Introduction of the construct into the hostcell can be effected by calcium phosphate transfection, DEAE-Dextranmediated transfection, lipofection or electroporation. (Davis, L.,Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).

[0048] The constructs in host cells can be used in a conventional mannerto produce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

[0049] Mature proteins can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., (1989), the disclosure of which is hereby incorporated byreference.

[0050] Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples including the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

[0051] Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

[0052] Useful expression vectors for bacterial use are constructed byinserting a structural DNA sequence encoding a desired protein togetherwith suitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

[0053] As a representative but nonlimiting example, useful expressionvectors for bacterial use can comprise a selectable marker and bacterialorigin of replication derived from commercially available plasmidscomprising genetic elements of the well known cloning vector pBR322(ATCC 37017). Such commercial vectors include, for example, pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec,Madison, Wis., USA). These pBR322 “backbone” sections are combined withan appropriate promoter and the structural sequence to be expressed.

[0054] Following transformation of a suitable host strain and growth ofthe host strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

[0055] Cells are typically harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification.

[0056] Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents, suchmethods are well know to those skilled in the art.

[0057] Various mammalian cell culture systems can also be employed toexpress recombinant protein. Examples of mammalian expression systemsinclude the COS-7 lines of monkey kidney fibroblasts, described byGluzman, Cell, 23:175 (1981), and other cell lines capable of expressinga compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors may comprise an origin ofreplication, a suitable promoter and enhancer, polyadenylation site,splice donor and acceptor sites, transcriptional termination sequences,and 5′ flanking nontranscribed sequences. DNA sequences derived from theSV40 splice and polyadenylation sites may be used to provide therequired nontranscribed genetic elements.

[0058] The ICE-LAP-3 and 4 polypeptides can be recovered and purifiedfrom recombinant cell cultures by methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography hydroxylapatite chromatographyand lectin chromatography. Protein refolding steps can be used, asnecessary, in completing configuration of the mature protein. Finally,high performance liquid chromatography (HPLC) can be employed for finalpurification steps.

[0059] The polypeptides of the present invention may be a naturallypurified product, or a product of chemical synthetic procedures, orproduced by recombinant techniques from a prokaryotic or eukaryotic host(for example, by bacterial, yeast, higher plant, insect and mammaliancells in culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue.

[0060] The ICE-LAP-3 and 4 polypeptides may be employed to treatabnormally controlled programmed cell death. Abnormally controlledprogrammed cell death may be an underlying cause of cancers due to anabnormal amount of cell growth. Accordingly, since ICE-LAP genes areimplicated in programmed cell death, they may be used to target unwantedcells, for example, cancerous cells. ICE-LAP-3 and 4 may also be used tocontrol vertebrate development and tissue homeostasis, due to itsapoptosis ability.

[0061] Also, ICE-LAP-3 and 4 polypeptides may be used to overcome manyviral infections by overcoming the suppressed programmed cell death,since programmed cell death may be one of the primary antiviral defensemechanisms of cells.

[0062] ICE-LAP-3 and 4 may also be employed to treat immuno-suppressionrelated disorders, such as AIDS, by targeting virus infected cells forcell death.

[0063] This invention also provides the use of the human ICE-LAP-3 and 4genes as a diagnostic. For example, some diseases result from inheriteddefective genes. These genes can be detected by comparing the sequenceof the defective gene with that of a normal one. That is, a mutant genewould be associated with abnormal cell growth, for example cancer, or asusceptibility to abnormal cell growth.

[0064] Individuals carrying mutations in the human ICE-LAP-3 and 4 genesmay be detected at the DNA level by a variety of techniques. Nucleicacids used for diagnosis may be obtained from a patient's cells, such asfrom blood, urine, saliva, tissue biopsy and autopsy material. Thegenomic DNA may be used directly for detection or may be amplifiedenzymatically by using PCR (Saiki et al., Nature, 324:163-166 (1986)prior to analysis. RNA or cDNA may also be used for the same purpose.Deletions or insertions can be detected by a change in size of theamplified product in comparison to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to radiolabeled ICE-LAP-3and 4 RNA or alternatively, radiolabeled ICE-LAP-3 and 4 antisense DNAsequences. Perfectly matched sequences can be distinguished frommismatched duplexes by RNase A digestion or by differences in meltingtemperatures.

[0065] Genetic testing based on DNA sequence differences may be achievedby detection of alteration in electrophoretic mobility of DNA fragmentsin gels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242 (1985)).

[0066] Sequence changes at specific locations may also be revealed bynuclease protection assays, such as RNase protection and S1 protectionor the chemical cleavage method (e.g., Cotton et al., PNAS, USA,85:4397-4401 (1985)).

[0067] Thus, the detection of a specific DNA sequence may be achieved bymethod such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing, or the use of restriction enzymes, e.g.,restriction fragment length polymorphisms, and Southern blotting ofgenomic DNA. Also, mutations may be detected by in situ analysis.

[0068] In addition, some diseases are a result of, or are characterizedby, changes in gene expression which can be detected by changes in themRNA. Alternatively, the ICE-LAP-3 and 4 genes can be used as areference to identify individuals expressing a decreased level ofICE-LAP-3 and 4 protein, e.g., by Northern blotting.

[0069] The present invention also relates to a diagnostic assay fordetecting levels of ICE-LAP-3 and 4 protein in a sample taken from ahost, for example, a blood, urine or serum sample. An altered level ofthe ICE-LAP polypeptides is indicative of a cancer or immunodeficiencydiagnosis. The level of ICE-LAP-3 and 4 may be detected, for example, byan immunoassay technique by procedures which are apparent to those ofskill in the art from the teachings herein. An example of such an assayis a sandwich assay which utilizes two antibodies specific to anICE-LAP-3 or 4 antigen, preferably monoclonal antibodies with one of theantibodies being labeled, eg. by coupling a suitable label such as anindicator enzyme, eg. horseradish peroxidase. The unlabeled antibody ispreferably on a solid support. If antigen is present, the antigen willbind to both antibodies. After binding of the peroxidase-coupledantibody to the antigen, the peroxidase can be used to generate acolored product that is measurable and whose concentration is related tothe amount of antigen in a sample. Because of the catalytic nature ofthe enzyme the system greatly amplifies the signal. Altered levels ofICE-LAP-3 and 4 are indicative of the particular diseases mentionedabove. Also, an ELISA assay may be employed to detect the amount ofICE-LAP-3 and 4 in a sample.

[0070] The polypeptides of the present invention may also be used foridentifying other molecules which have similar biological activity. Anexample of a screen for this comprises isolating the coding region ofthe ICE-LAP genes by using the known DNA sequence to synthesize anoligonucleotide probe. Labeled oligonucleotides having a sequencecomplementary to that of the gene of the present invention are used toscreen a library of human cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

[0071] The present invention is further related to a process ofscreening molecules to identify antagonists or agonists of the ICE-LAP-3and 4 polypeptides of the present invention. Agonists increase thenatural biological function of ICE-LAP-3 and 4, while antagonists reduceor eliminate such function. An example of such an assay comprisescombining ICE-LAP-3 and 4 and a potential antagonist or agonist compoundwith their natural substrate under conditions allowing for action uponthe substrate and determining whether the compound prevents ICE-LAP-3 or4 from cleaving the substrate or enhances the cleavage.

[0072] Potential antagonists include an antibody, or in some cases, anoligonucleotide, which binds to the polypeptide. Alternatively, apotential antagonist may be a closely related protein which binds to thesubstrate, however, they are inactive forms of the polypeptide andthereby prevent the action of ICE-LAP-3 and 4.

[0073] Another potential antagonist is an antisense construct preparedusing antisense technology. Antisense technology can be used to controlgene expression through triple-helix formation or antisense DNA or RNA,both of which methods are based on binding of a polynucleotide to DNA orRNA. For example, the 5′ coding portion of the polynucleotide sequence,which encodes for the mature polypeptides of the present invention, isused to design an antisense RNA oligonucleotide of from about 10 to 40base pairs in length. A DNA oligonucleotide is designed to becomplementary to a region of the gene involved in transcription (triplehelix—see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al,Science, 241:456 (1988); and Dervan et al., Science, 251: 1360 (1991)),thereby preventing transcription and the production of ICE-LAP-3 and 4.The antisense RNA oligonucleotide hybridizes to the mRNA in vivo andblocks translation of the mRNA molecule into the polypeptides(antisense—Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides asAntisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988)). The oligonucleotides described above can also be delivered tocells such that the antisense RNA or DNA may be expressed in vivo toinhibit production of ICE-LAP-3 and 4.

[0074] Potential antagonists include a small molecule which binds to andoccupies the catalytic site of the polypeptide thereby making thecatalytic site inaccessible to substrate such that normal biologicalactivity is prevented. Examples of small molecules include but are notlimited to small peptides or peptide-like molecules.

[0075] The antagonists may be employed to treat non-programmed necroticcell death related to cardiovascular diseases, strokes, trauma, andother degenerative diseases where abnormal regulation of ICE-LAP-3 and 4may lead to pathological cell death, for example,immunosuppression-related disorders, Alzheimer's disease, Parkinson'sdisease, rheumatoid arthritis.

[0076] The antagonists may also be used to treat immune-based diseasesof the lung and airways, central nervous system, eyes and ears, joints,bones, cardiovascular system and gastrointestinal and urogenitalsystems. The antagonists may be employed in a composition with apharmaceutically acceptable carrier, e.g., as hereinafter described.

[0077] The polypeptides and antagonists and agonists of the presentinvention may be employed in combination with a suitable pharmaceuticalcarrier. Such compositions comprise a therapeutically effective amountof the polypeptide or antagonist or agonist, and a pharmaceuticallyacceptable carrier or excipient. Such a carrier includes but is notlimited to saline, buffered saline, dextrose, water, glycerol, ethanol,and combinations thereof. The formulation should suit the mode ofadministration.

[0078] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. Inaddition, the polypeptides of the present invention may be employed inconjunction with other therapeutic compounds.

[0079] The pharmaceutical compositions may be administered in aconvenient manner such as by the intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal or intradermal routes. Thepharmaceutical compositions are administered in an amount which iseffective for treating and/or prophylaxis of the specific indication. Ingeneral, they will be administered in an amount of at least 10 μg/kgbody weight, and in most cases they will be administered in an amountnot in excess of 8 mg/Kg body weight per day. In most cases, the dosageis from about 10 μg/kg to about 1 mg/kg body weight daily, taking intoaccount the routes of administration, symptoms, etc.

[0080] The ICE-LAP-3 and 4 polypeptides, and agonists and antagonistswhich are polypeptides may also be employed in accordance with thepresent invention by expression of such polypeptides in vivo, which isoften referred to as “gene therapy.”

[0081] Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art. For example, cellsmay be engineered by procedures known in the art by use of a retroviralparticle containing RNA encoding a polypeptide of the present invention.

[0082] Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Asknown in the art, a producer cell for producing a retroviral particlecontaining RNA encoding the polypeptide of the present invention may beadministered to a patient for engineering cells in vivo and expressionof the polypeptide in vivo. These and other methods for administering apolypeptide of the present invention by such method should be apparentto those skilled in the art from the teachings of the present invention.For example, the expression vehicle for engineering cells may be otherthan a retrovirus, for example, an adenovirus which may be used toengineer cells in vivo after combination with a suitable deliveryvehicle.

[0083] The sequences of the present invention are also valuable forchromosome identification. The sequence is specifically targeted to andcan hybridize with a particular location on an individual humanchromosome. Moreover, there is a current need for identifying particularsites on the chromosome. Few chromosome marking reagents based on actualsequence data (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

[0084] Briefly, sequences can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp) from the cDNA. Computer analysis of thecDNA is used to rapidly select primers that do not span more than oneexon in the genomic DNA, thus complicating the amplification process.These primers are then used for PCR screening of somatic cell hybridscontaining individual human chromosomes. Only those hybrids containingthe human gene corresponding to the primer will yield an amplifiedfragment.

[0085] PCR mapping of somatic cell hybrids is a rapid procedure forassigning a particular DNA to a particular chromosome. Using the presentinvention with the same oligonucleotide primers, sublocalization can beachieved with panels of fragments from specific chromosomes or pools oflarge genomic clones in an analogous manner. Other mapping strategiesthat can similarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

[0086] Fluorescence in situ hybridization (FISH) of a cDNA clones to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 500 or 600 bases; however, clones larger than 2,000 bp havea higher likelihood of binding to a unique chromosomal location withsufficient signal intensity for simple detection. FISH requires use ofthe clones from which the EST was derived, and the longer the better.For example, 2,000 bp is good, 4,000 is better, and more than 4,000 isprobably not necessary to get good results a reasonable percentage ofthe time. For a review of this technique, see Verma et al., HumanChromosomes: a Manual of Basic Techniques, Pergamon Press, New York(1988).

[0087] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. Such data are found, for example, inV. McKusick, Mendelian Inheritance in Man (available on line throughJohns Hopkins University Welch Medical Library). The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion are then identified through linkage analysis (coinheritance ofphysically adjacent genes).

[0088] Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

[0089] With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes (this assumes 1 megabase mapping resolution and one geneper 20 kb).

[0090] The polypeptides, their fragments or other derivatives, oranalogs thereof, or cells expressing them can be used as an immunogen toproduce antibodies thereto. These antibodies can be, for example,polyclonal or monoclonal antibodies. The present invention also includeschimeric, single chain, and humanized antibodies, as well as Fabfragments, or the product of an Fab expression library. Variousprocedures known in the art may be used for the production of suchantibodies and fragments.

[0091] Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

[0092] For preparation of monoclonal antibodies, any technique whichprovides antibodies produced by continuous cell line cultures can beused. Examples include the hybridoma technique (Kohler and Milstein,1975, Nature, 256:495-497), the trioma technique, the human B-cellhybridoma-technique (Kozbor et al., 1983, Immunology Today 4:72), andthe EBV-hybridoma technique to produce human monoclonal antibodies(Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, Inc., pp. 77-96).

[0093] Techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produce singlechain antibodies to immunogenic polypeptide products of this invention.

[0094] The present invention will be further described with reference tothe following examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

[0095] In order to facilitate understanding of the following examplescertain frequently occurring methods and/or terms will be described.

[0096] “Plasmids” are designated by a lower case p preceded and/orfollowed by capital letters and/or numbers. The starting plasmids hereinare either commercially available, publicly available on an unrestrictedbasis, or can be constructed from available plasmids in accord withpublished procedures. In addition, equivalent plasmids to thosedescribed are known in the art and will be apparent to the ordinarilyskilled artisan.

[0097] “Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

[0098] Size separation of the cleaved fragments is performed using 8percent polyacrylamide gel described by Goeddel, D. et al., NucleicAcids Res., 8:4057 (1980), or agarose gels (0.5-1.5%).

[0099] “Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

[0100] “Ligation ” refers to the process of forming phosphodiester bondsbetween two double stranded nucleic acid fragments (Maniatis, T., etal., Id., p. 146). Unless otherwise provided, ligation may beaccomplished using known buffers and conditions with 10 units to T4 DNAligase (“ligase”) per 0.5 μg of approximately equimolar amounts of theDNA fragments to be ligated.

[0101] Unless otherwise stated, transformation was performed asdescribed in the method of Graham, F. and Van der Eb, A., Virology,52:456-457 (1973).

EXAMPLE 1

[0102] Bacterial Expression and Purification of ICE-LAP-3

[0103] The DNA sequence encoding ICE-LAP-3, ATCC # 75875, is initiallyamplified using PCR oligonucleotide primers corresponding to the 5′sequences of the processed ICE-LAP-3 protein (minus the signal peptidesequence) and the vector sequences 3′ to the ICE-LAP-3 gene. Additionalnucleotides corresponding to ICE-LAP-3 are added to the 5′ and 3′sequences respectively. The 5′ oligonucleotide primer has the sequence5′ GATCGGATCCATGCGTGCGGGGACACGGGTC 3′ contains a Bam HI restrictionenzyme site (underlined) followed by 18 nucleotides of ICE-LAP-3 codingsequence starting from the presumed terminal amino acid of the processedprotein codon. The 3′ sequence 5′ GTACTCTAGATCATTCACCCTGGTGGAGGAT 3′contains complementary sequences to an Xba I site (underlined) followedby 21 nucleotides of ICE-LAP-3. The restriction enzyme sites correspondto the restriction enzyme sites on the bacterial expression vector pQE-9(Qiagen, Inc. 9259 Eton Avenue, Chatsworth, Calif., 91311). pQE-9encodes antibiotic resistance. (Amp^(r)), a bacterial origin ofreplication (ori), an IPTG-regulatable promoter operator (P/O), aribosome binding site (RBS), a 6-His tag and restriction enzyme sites.pQE-9 is then digested with Bam HI and Xba I. The amplified sequencesare ligated into pQE-9 and are inserted in frame with the sequenceencoding for the histidine tag and the RBS. The ligation mixture is thenused to transform E. coli available from Qiagen under the trademarkM15/rep 4 by the procedure described in Sambrook, J. et al., MolecularCloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989).M15/rep4 contains multiple copies of the plasmid pREP4, which expressesthe lacI repressor and also confers kanamycin resistance (Kan^(r)).Transformants are identified by their ability to grow on LB plates andampicillin/kanamycin resistant colonies are selected. Plasmid DNA isisolated and confirmed by restriction analysis. Clones containing thedesired constructs are grown overnight (O/N) in liquid culture in LBmedia supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/Nculture is used to inoculate a large culture at a ratio of 1:100 to1:250. The cells are grown to an optical density 600 (O.D.⁶⁰⁰) ofbetween 0.4 and 0.6. IPTG (“Isopropyl-B-D-thiogalacto pyranoside”) isthen added to a final concentration of 1 mM. IPTG induces byinactivating the lacI repressor, clearing the P/O leading to increasedgene expression. Cells are grown an extra 3 to 4 hours. Cells are thenharvested by centrifugation. The cell pellet is solubilized in thechaotropic agent 6 Molar Guanidine HCl. After clarification, solubilizedICE-LAP-3 is purified from this solution by chromatography on aNickel-Chelate column under conditions that allow for tight binding byproteins containing the 6-His tag (Hochuli, E. et al., J. Chromatography411:177-184 (1984)). ICE-LAP-3 (95% pure is eluted from the column in 6molar guanidine HCl pH 5.0 and for the purpose of renaturation adjustedto 3 molar guanidine HCl, 100 mM sodium phosphate, 10 mmolar glutathione(reduced) and 2 mmolar glutathione (oxidized). After incubation in thissolution for 12 hours the protein was dialyzed to 10 mmolar sodiumphosphate.

EXAMPLE 2

[0104] Bacterial Expression and Purification of ICE-LAP-4

[0105] The DNA sequence encoding ICE-LAP-4, ATCC # 75873, is initiallyamplified using PCR oligonucleotide primers corresponding to the 5′sequences of the processed ICE-LAP-4 protein (minus the signal peptidesequence) and the vector sequences 3′ to the ICE-LAP-4 gene. Additionalnucleotides corresponding to ICE-LAP-4 are added to the 5′ and 3′sequences respectively. The 5′ oligonucleotide primer has the sequence5′ GATCGGATCCATGGAGAACACTGAAAACTCA 3′ contains a Bam HI restrictionenzyme site (underlined) followed by 18 nucleotides of ICE-LAP-4 codingsequence starting from the presumed terminal amino acid of the processedprotein codon. The 3′ sequence 5′ GTACTCTAGATTAGTGATAAAAATAGAGTTC 3′contains complementary sequences to an Xba I site (underlined) followedby 21 nucleotides of ICE-LAP-4. The restriction enzyme sites correspondto the restriction enzyme sites on the bacterial expression vector pQE-9(Qiagen, Inc. 9259 Eton Avenue, Chatsworth, Calif., 91311). pQE-9encodes antibiotic resistance (Amp^(r)), a bacterial origin ofreplication (ori), an IPTG-regulatable promoter operator (P/O), aribosome binding site (RBS), a 6-His tag and restriction enzyme sites.pQE-9 is then digested with Bam HI and Xba I. The amplified sequencesare ligated into pQE-9 and are inserted in frame with the sequenceencoding for the histidine tag and the RBS. The ligation mixture is thenused to transform E. coli available from Qiagen under the trademarkM15/rep 4 by the procedure described in Sambrook, J. et al., MolecularCloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989).M15/rep4 contains multiple copies of the plasmid pREP4, which expressesthe lacI repressor and also confers kanamycin resistance (Kan^(r)).Transformants are identified by their ability to grow on LB plates andampicillin/kanamycin resistant colonies are selected. Plasmid DNA isisolated and confirmed by restriction analysis. Clones containing thedesired constructs are grown overnight (O/N) in liquid culture in LBmedia supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/Nculture is used to inoculate a large culture at a ratio of 1:100 to1:250. The cells are grown to an optical density 600 (O.D.⁶⁰⁰) ofbetween 0.4 and 0.6. IPTG (“Isopropyl-B-D-thiogalacto pyranoside”) isthen added to a final concentration of 1 mM. IPTG induces byinactivating the lacI repressor, clearing the P/O leading to increasedgene expression. Cells are grown an extra 3 to 4 hours. Cells are thenharvested by centrifugation. The cell pellet is solubilized in thechaotropic agent 6 Molar Guanidine HCl. After clarification, solubilizedICE-LAP-4 is purified from this solution by chromatography on aNickel-Chelate column under conditions that allow for tight binding byproteins containing the 6-His tag (Hochuli, E. et al., J. Chromatography411:177-184 (1984)). ICE-LAP-4 (95% pure is eluted from the column in 6molar guanidine HCl pH 5.0 and for the purpose of renaturation adjustedto 3 molar guanidine HCl, 100 mM sodium phosphate, 10 mmolar glutathione(reduced) and 2 mmolar glutathione (oxidized). After incubation in thissolution for 12 hours the protein was dialyzed to 10 mmolar sodiumphosphate.

EXAMPLE 3

[0106] Expression of Recombinant ICE-LAP-3 in COS Cells

[0107] The expression of a plasmid, ICE-LAP-3 HA, is derived from avector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin ofreplication, 2) ampicillin resistance gene, 3) E.coli replicationorigin, 4) CMV promoter followed by a polylinker region, a SV40 intronand polyadenylation site. A DNA fragment encoding the entire ICE-LAP-3precursor and a HA tag fused in frame to its 3′ end was cloned into thepolylinker region of the vector, therefore, the recombinant proteinexpression is directed under the CMV promoter. The HA tag correspond toan epitope derived from the influenza hemagglutinin protein aspreviously described (I. Wilson, H. Niman, R. Heighten, A Cherenson, M.Connolly, and R. Lerner, 1984, Cell 37, 767). The infusion of HA tag toour target protein allows easy detection of the recombinant protein withan antibody that recognizes the HA epitope.

[0108] The plasmid construction strategy is described as follows:

[0109] The DNA sequence encoding for ICE-LAP-3, ATCC # 75875, wasconstructed by PCR on the full-length ICE-LAP-3 using two primers: the5′ primer 5′ GACTATGCGTGCGGGGACACGG 3′ contains the ICE-LAP-3translational initiation site ATG followed by 5 nucleotides of ICE-LAP-3coding sequence starting from the initiation codon; the 3′ sequence 5′AATCAAGCGTAG TCTGGGACGTCGTATGGGTATTCACCCTGGTGGAGGATTTG 3′ containstranslation stop codon, HA tag and the last 21 nucleotides of theICE-LAP-3 coding sequence (not including the stop codon). Therefore, thePCR product contains the ICE-LAP-3 coding sequence followed by HA tagfused in frame, and a translation termination stop codon next to the HAtag. The PCR amplified DNA fragment was ligated with pcDNAI/Amp by bluntend ligation. The ligation mixture was transformed into E. coli strainSURE (available from Stratagene Cloning Systems, 11099 North TorreyPines Road, La Jolla, Calif. 92037) the transformed culture was platedon ampicillin media plates and resistant colonies were selected. PlasmidDNA was isolated from transformants and examined by restriction analysisfor the presence of the correct fragment. For expression of therecombinant ICE-LAP-3, COS cells were transfected with the expressionvector by DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press,(1989)). The expression of the ICE-LAP-3 HA protein was detected byradiolabelling and immunoprecipitation method (E. Harlow, D. Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,(1988)). Cells were labelled for 8 hours with ³⁵S-cysteine two days posttransfection. Culture media were then collected and cells were lysedwith detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40,0.5% DOC, 50mM Tris, pH 7.5) (Wilson, I. et al., Id. 37:767 (1984)).Both cell lysate and culture media were precipitated with a HA specificmonoclonal antibody. Proteins precipitated were analyzed on 15% SDS-PAGEgels.

EXAMPLE 4

[0110] Expression of Recombinant ICE-LAP-4 in COS Cells

[0111] The expression of a plasmid, ICE-LAP-4 HA, is derived from avector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin ofreplication, 2) ampicillin resistance gene, 3) E. coli replicationorigin, 4) CMV promoter followed by a polylinker region, a SV40 intronand polyadenylation site. A DNA fragment encoding the entire ICE-LAP-4precursor and a HA tag fused in frame to its 3′ end was cloned into thepolylinker region of the vector, therefore, the recombinant proteinexpression is directed under the CMV promoter. The HA tag correspond toan epitope derived from the influenza hemagglutinin protein aspreviously described (I. Wilson, H. Niman, R. Heighten, A Cherenson, M.Connolly, and R. Lerner, 1984, Cell 37, 767). The infusion of HA tag toour target protein allows easy detection of the recombinant protein withan antibody that recognizes the HA epitope.

[0112] The plasmid construction strategy is described as follows:

[0113] The DNA sequence encoding for ICE-LAP-4, ATCC # 75873, wasconstructed by PCR on the full-length ICE-LAP-4 using two primers: the5′ primer 5′ ACCATGGAGAACACTGAAAAC 3′ contains the ICE-LAP-4translational initiation site, ATG, followed by 15 nucleotides ofICE-LAP-4 coding sequence starting from the initiation codon; the 3′sequence 5′ AATCAAGCGTAGTCTGGGACGTCGTATGGGTAGTGATAAAAATAGAGTTCTTT 3′contains translation stop codon, HA tag and the last 21 nucleotides ofthe ICE-LAP-4 coding sequence (not including the stop codon). Therefore,the PCR product contains the ICE-LAP-4 coding sequence followed by HAtag fused in frame, and a translation termination stop codon next to theHA tag. The PCR amplified DNA fragment was ligated with pcDNAI/Amp byblunt end ligation. The ligation mixture was transformed into E. colistrain SURE (available from Stratagene Cloning Systems, 11099 NorthTorrey Pines Road, La Jolla, Calif. 92037) the transformed culture wasplated on ampicillin media plates and resistant colonies were selected.Plasmid DNA was isolated from transformants and examined by restrictionanalysis for the presence of the correct fragment. For expression of therecombinant ICE-LAP-4, COS cells were transfected with the expressionvector by the DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press,(1989)). The expression of the ICE-LAP-4 HA protein was detected byradiolabelling and immunoprecipitation method (E. Harlow, D. Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,(1988)). Cells were labelled for 8 hours with ³⁵S-cysteine two days posttransfection. Culture media were then collected and cells were lysedwith detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40,0.5% DOC, 50 mM Tris, pH 7.5) (Wilson, I. et al., Id. 37:767 (1984)).Both cell lysate and culture media were precipitated with a HA specificmonoclonal antibody. Proteins precipitated were analyzed on 15% SDS-PAGEgels.

EXAMPLE 5

[0114] Expression Pattern of ICE-LAP-3 in Human Tissue

[0115] Northern blot analysis was carried out to examine the levels ofexpression of ICE-LAP-3 in human tissues. Total cellular RNA sampleswere isolated with RNAzol™ B system (Biotecx Laboratories, Inc. 6023South Loop East, Houston, Tex. 77033). About 10 μg of total RNA isolatedfrom each human tissue specified was separated on 1% agarose gel andblotted onto a nylon filter (Sambrook, Fritsch, and Maniatis, MolecularCloning, Cold Spring Harbor Press, (1989)). The labeling reaction wasdone according to the Stratagene Prime-It kit with 50 ng DNA fragment.The labeled DNA was purified with a Select-G-50 column. (5 Prime—3Prime, Inc. 5603 Arapahoe Road, Boulder, Colo. 80303). The filter wasthen hybridized with radioactive labeled full length ICE-LAP-3 gene at1,000,000 cpm/ml in 0.5 M NaPO₄, pH 7.4 and 7% SDS overnight 65° C.After wash twice at room temperature and twice at 60° C. with 0.5×SSC,0.1% SDS, the filter was then exposed at −70° C. overnight with anintensifying screen. The message RNA for ICE-LAP-3 is abundant in liver.

EXAMPLE 6

[0116] Expression Pattern of ICE-LAP-4 in Human Tissue

[0117] Northern blot analysis is carried out to examine the levels ofexpression of ICE-LAP-4 in human tissues. Total cellular RNA sampleswere isolated with RNAzol™ B system (Biotecx Laboratories, Inc. 6023South Loop East, Houston, Tex. 77033). About 10 μg of total RNA isolatedfrom each human tissue specified was separated on 1% agarose gel andblotted onto a nylon filter (Sambrook, Fritsch, and Maniatis, MolecularCloning, Cold Spring Harbor Press, (1989)). The labeling reaction wasdone according to the Stratagene Prime-It kit with 50 ng DNA fragment.The labeled DNA was purified with a Select-G-50 column. (5 Prime—3Prime, Inc. 5603 Arapahoe Road, Boulder, Colo. 80303). The filter wasthen hybridized with radioactive labeled full length ICE-LAP-4 gene at1,000,000 cpm/ml in 0.5 M NaPO₄, pH 7.4 and 7% SDS overnight at 65° C.After wash twice at room temperature and twice at 60° C. with 0.5×SSC,0.1% SDS, the filter was then exposed at −70° C. overnight with anintensifying screen.

[0118] Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

1 12 1369 base pairs nucleic acid single linear DNA (genomic) 1GCACGAGAAA CTTTGCTGTG CGCGTTCTCC CGCGCGCGGG CTCAACTTTG TAGAGCGAGG 60GGCCAACTTG GCAGAGCGCG CGGCCAGCTT TGCAGAGAGC GCCCTCCAGG GACTATGCG 120GCGGGGACAC GGGTCGCTTT GGGCTCTTCC ACCCCTGCGG AGCGCACTAC CCCGAGCCA 180GGGCGGTGCA AGCCCCGCCC GGCCCTACCC AGGGCGGCTC CTCCCTCCGC AGCGCCGAG 240CTTTTAGTTT CGCTTTCGCT AAAGGGGCCC CAGACCCTTG CTGCGGAGCG ACGGAGAGA 300ACTGTGCCAG TCCCAGCCGC CCTACCGCCG TGGGAACGAT GGCAGATGAT CAGGGCTGT 360TTGAAGAGCA GGGGGTTGAG GATTCAGCAA ATGAAGATTC AGTGGATGCT AAGCCAGAC 420GGTCCTCGTT TGTACCGTCC CTCTTCAGTA AGAAGAAGAA AAATGTCACC ATGCGATCC 480TCAAGACCAC CCGGGACCGA GTGCCTACAT ATCAGTACAA CATGAATTTT GAAAAGCTG 540GCAAATGCAT CATAATAAAC AACAAGAACT TTGATAAAGT GACAGGTATG GGCGTTCGA 600ACGGAACAGA CAAAGATGCC GAGGCGCTCT TCAAGTGCTT CCGAAGCCTG GGTTTTGAC 660TGATTGTCTA TAATGACTGC TCTTGTGCCA AGATGCAAGA TCTGCTTAAA AAAGCTTCT 720AAGAGGACCA TACAAATGCC GCCTGCTTCG CCTGCATCCT CTTAAGCCAT GGAGAAGAA 780ATGTAATTTA TGGGAAAGAT GGTGTCACAC CAATAAAGGA TTTGACAGCC CACTTTAGG 840GGGATAGATG CAAAACCCTT TTAGAGAAAC CCAAACTCTT CTTCATTCAG GCTTGCCGA 900GGACCGAGCT TGATGATGCC ATCCAGGCCG ACTCGGGGCC CATCAATGAC ACAGATGCT 960ATCCTCGATA CAAGATCCCA GTGGAAGCTG ACTTCCTCTT CGCCTATTCC ACGGTTCC 1020GCTATTACTC GTGGAGGAGC CCAGGAAGAG GCTCCTGGTT TGTGCAAGCC CTCTGCTC 1080TCCTGGAGGA GCACGGAAAA GAGCTGGAAA TCATGCAAAT CCTCACCAGG GTGAATGA 1140GAGTTGCCAG GCACTTTGAG TCTCAGTCTG ATGACCCACA CTTCCATGAG AAGAAGCA 1200TCCCCTGTGT GGTCTCCATG CTCACCAAGG AACTCTACTT CAGTCAATAG CCATATCA 1260GGTACATTCT AGCTGAGAAG CAATGGGTCA CTCATTAATG AATCACATTT TTTTATGC 1320TTGAAATATT CAGAAATTCT CCAGGATTTT AATTTCAGGA AAATGTATT 1369 303 aminoacids amino acid single linear protein 2 Met Ala Asp Asp Gln Gly Cys IleGlu Glu Gln Gly Val Glu Asp Se 1 5 10 15 Ala Asn Glu Asp Ser Val Asp AlaLys Pro Asp Arg Ser Ser Phe Va 20 25 30 Pro Ser Leu Phe Ser Lys Lys LysLys Asn Val Thr Met Arg Ser Il 35 40 45 Lys Thr Thr Arg Asp Arg Val ProThr Tyr Gln Tyr Asn Met Asn Ph 50 55 60 Glu Lys Leu Gly Lys Cys Ile IleIle Asn Asn Lys Asn Phe Asp Ly 65 70 75 80 Val Thr Gly Met Gly Val ArgAsn Gly Thr Asp Lys Asp Ala Glu Al 85 90 95 Leu Phe Lys Cys Phe Arg SerLeu Gly Phe Asp Val Ile Val Tyr As 100 105 110 Asp Cys Ser Cys Ala LysMet Gln Asp Leu Leu Lys Lys Ala Ser Gl 115 120 125 Glu Asp His Thr AsnAla Ala Cys Phe Ala Cys Ile Leu Leu Ser Hi 130 135 140 Gly Glu Glu AsnVal Ile Tyr Gly Lys Asp Gly Val Thr Pro Ile Ly 145 150 155 160 Asp LeuThr Ala His Phe Arg Gly Asp Arg Cys Lys Thr Leu Leu Gl 165 170 175 LysPro Lys Leu Phe Phe Ile Gln Ala Cys Arg Gly Thr Glu Leu As 180 185 190Asp Ala Ile Gln Ala Asp Ser Gly Pro Ile Asn Asp Thr Asp Ala As 195 200205 Pro Arg Tyr Lys Ile Pro Val Glu Ala Asp Phe Leu Phe Ala Tyr Se 210215 220 Thr Val Pro Gly Tyr Tyr Ser Trp Arg Ser Pro Gly Arg Gly Ser Tr225 230 235 240 Phe Val Gln Ala Leu Cys Ser Ile Leu Glu Glu His Gly LysGlu Le 245 250 255 Glu Ile Met Gln Ile Leu Thr Arg Val Asn Asp Arg ValAla Arg Hi 260 265 270 Phe Glu Ser Gln Ser Asp Asp Pro His Phe His GluLys Lys Gln Il 275 280 285 Pro Cys Val Val Ser Met Leu Thr Lys Glu LeuTyr Phe Ser Gln 290 295 300 1159 base pairs nucleic acid single linearDNA (genomic) 3 GCACGAGCGG ATGGGTGCTA TTGTGAGGCG GTTGTAGAAG AGTTTCGTGAGTGCTCGCAG 60 CTCATACCTG TGGCTGTGTA TCCGTGGCCA CAGCTGGTTG GCGTCGCCTTGAAATCCCA 120 GCCGTGAGGA GTTAGCGAGC CCTGCTCACA CTCGGCGCTC TGGTTTTCGGTGGGTGTGC 180 CTGCACCTGC CTCTTCCCGC ATTCTCATTA ATAAAGGTAT CCATGGAGAACACTGAAAA 240 TCAGTGGATT CAAAATCCAT TAAAAATTTG GAACCAAAGA TCATACATGGAAGCGAATC 300 ATGGACTCTG GAATATCCCT GGACAACAGT TATAAAATGG ATTATCCTGAGATGGGTTT 360 TGTATAATAA TTAATAATAA GAATTTTCAT AAAAGCACTG GAATGACATCTCGGTCTGG 420 ACAGATGTCG ATGCAGCAAA CCTCAGGGAA ACATTCAGAA ACTTGAAATATGAAGTCAG 480 AATAAAAATG ATCTTACACG TGAAGAAATT GTGGAATTGA TGCGTGATGTTTCTAAAGA 540 GATCACAGCA AAAGGAGCAG TTTTGTTTGT GTGCTTCTGA GCCATGGTGAAGAAGGAAT 600 ATTTTTGGAA CAAATGGACC TGTTGACCTG AAAAAAATAA CAAACTTTTTCAGAGGGGA 660 CGTTGTAGAA GTCTAACTGG AAAACCCAAA CTTTTCATTA TTCAGGCCTGCCGTGGTAC 720 GAACTGGACT GTGGCATTGA GACAGACAGT GGTGTTGATG ATGACATGGCGTGTCATAA 780 ATACCAGTGG AGGCCGACTT CTTGTATGCA TACTCCACAG CACCTGGTTATTATTCTTG 840 CGAAATTCAA AGGATGGCTC CTGGTTCATC CAGTCGCTTT GTGCCATGCTGAAACAGTA 900 GCCGACAAGC TTGAATTTAT GCACATTCTT ACCCGGGTTA ACCGAAAGGTGGCAACAGA 960 TTTGAGTCCT TTTCCTTTGA CGCTACTTTT CATGCAAAGA AACAGATTCCATGTATTG 1020 TCCATGCTCA CAAAAGAACT CTATTTTTAT CACTAAAGAA ATGGTTGGTTGGTGGTTT 1080 TTTAGTTTGT ATGCCAAGTG AGAAGATGGT ATATTTGGGT ACTGTATTTCCCTCTCAT 1140 GGGACCTACT CTCATGCTG 1159 277 amino acids amino acidsingle linear protein 4 Met Glu Asn Thr Glu Asn Ser Val Asp Ser Lys SerIle Lys Asn Le 1 5 10 15 Glu Pro Lys Ile Ile His Gly Ser Glu Ser Met AspSer Gly Ile Se 20 25 30 Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu MetGly Leu Cys Il 35 40 45 Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr GlyMet Thr Ser Ar 50 55 60 Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg GluThr Phe Arg As 65 70 75 80 Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp LeuThr Arg Glu Glu Il 85 90 95 Val Glu Leu Met Arg Asp Val Ser Lys Glu AspHis Ser Lys Arg Se 100 105 110 Ser Phe Val Cys Val Leu Leu Ser His GlyGlu Glu Gly Ile Ile Ph 115 120 125 Gly Thr Asn Gly Pro Val Asp Leu LysLys Ile Thr Asn Phe Phe Ar 130 135 140 Gly Asp Arg Cys Arg Ser Leu ThrGly Lys Pro Lys Leu Phe Ile Il 145 150 155 160 Gln Ala Cys Arg Gly ThrGlu Leu Asp Cys Gly Ile Glu Thr Asp Se 165 170 175 Gly Val Asp Asp AspMet Ala Cys His Lys Ile Pro Val Glu Ala As 180 185 190 Phe Leu Tyr AlaTyr Ser Thr Ala Pro Gly Tyr Tyr Ser Trp Arg As 195 200 205 Ser Lys AspGly Ser Trp Phe Ile Gln Ser Leu Cys Ala Met Leu Ly 210 215 220 Gln TyrAla Asp Lys Leu Glu Phe Met His Ile Leu Thr Arg Val As 225 230 235 240Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Ser Phe Asp Ala Thr Ph 245 250255 His Ala Lys Lys Gln Ile Pro Cys Ile Val Ser Met Leu Thr Lys Gl 260265 270 Leu Tyr Phe Tyr His 275 31 base pairs nucleic acid single linearDNA (genomic) 5 GATCGGATCC ATGCGTGCGG GGACACGGGT C 31 31 base pairsnucleic acid single linear DNA (genomic) 6 GTACTCTAGA TCATTCACCCTGGTGGAGGA T 31 31 base pairs nucleic acid single linear DNA (genomic) 7GATCGGATCC ATGGAGAACA CTGAAAACTC A 31 31 base pairs nucleic acid singlelinear DNA (genomic) 8 GTACTCTAGA TTAGTGATAA AAATAGAGTT C 31 22 basepairs nucleic acid single linear DNA (genomic) 9 GACTATGCGT GCGGGGACACGG 22 53 base pairs nucleic acid single linear DNA (genomic) 10AATCAAGCGT AGTCTGGGAC GTCGTATGGG TATTCACCCT GGTGGAGGAT TTG 53 21 basepairs nucleic acid single linear DNA (genomic) 11 ACCATGGAGA ACACTGAAAAC 21 53 base pairs nucleic acid single linear DNA (genomic) 12AATCAAGCGT AGTCTGGGAC GTCGTATGGG TAGTGATAAA AATAGAGTTC TTT 53

What is claimed is:
 1. An isolated polynucleotide selected from thegroup consisting of: (a) a polynucleotide encoding a polypeptide havingthe deduced amino acid sequence of FIG. 1 or a fragment, analog orderivative of said polypeptide; (b) a polynucleotide encoding apolypeptide having the deduced amino acid sequence of FIG. 2 or afragment, analog or derivative of said polypeptide; (c) a polynucleotideencoding a polypeptide having the amino acid sequence encoded by thecDNA contained in ATCC Deposit No. 75875 or a fragment, analog orderivative of said polypeptide; and (d) a polynucleotide encoding apolypeptide having the amino acid sequence encoded by the cDNA containedin ATCC Deposit No. 75873 or a fragment, analog or derivative of saidpolypeptide.
 2. The polynucleotides of claim 1 wherein thepolynucleotides are DNA.
 3. The polynucleotides of claim 1 wherein thepolynucleotides are RNA.
 4. The polynucleotides of claim 1 wherein thepolynucleotides are genomic DNA.
 5. A polynucleotide of claim 2 whereinsaid polynucleotide encodes a polypeptide having the deduced amino acidsequence of FIG.
 1. 6. A polynucleotide of claim 2 wherein saidpolynucleotide encodes a polypeptide having the deduced amino acidsequence of FIG.
 2. 7. A polynucleotide of claim 2 wherein saidpolynucleotide encode the polypeptide encoded by the cDNA of ATCCDeposit No.
 75875. 8. A polynucleotide of claim 2 wherein saidpolynucleotide encodes the polypeptide encoded by the CDNA of ATCCDeposit No.
 75873. 9. A polynucleotide of claim 1 having the codingsequence as shown in FIG.
 1. 10. A polynucleotide of claim 1 having thecoding sequence as shown in FIG.
 2. 11. A polynucleotide of claim 2having the coding sequence deposited as ATCC Deposit No.
 75875. 12. Apolynucleotide of claim 2 having the coding sequence deposited as ATCCDeposit No.
 75873. 13. A vector containing the DNA of claim
 2. 14. Ahost cell genetically engineered with the vector of claim
 13. 15. Aprocess for producing a polypeptide comprising: expressing from the hostcell of claim 14 the polypeptide encoded by said DNA.
 16. A process forproducing cells capable of expressing a polypeptide comprisinggenetically engineering cells with the vector of claim
 13. 17. Anisolated DNA hybridizable to the DNA of claim 2 and encoding apolypeptide having ICE-LAP-3 activity.
 18. An isolated DNA hybridizableto the DNA of claim 2 and encoding a polypeptide having ICE-LAP-4activity.
 19. A polypeptide selected from the group consisting of (i) apolypeptide having the deduced amino acid sequence of FIG. 1 andfragments, analogs and derivatives thereof; (ii) a polypeptide havingthe deduced amino acid sequence of FIG. 2 and fragments, analogs andderivatives thereof; (iii) a polypeptide encoded by the cDNA of ATCCDeposit No. 75875 and fragments, analogs and derivatives of saidpolypeptide; and (iv) a polypeptide encoded by the cDNA of ATCC DepositNo. 75873 and fragments, analogs and derivatives of said polypeptide.20. A polypeptide of claim 19 wherein the polypeptide has the deducedamino acid sequence of FIG.
 1. 21. A polypeptide of claim 19 wherein thepolypeptide has the deduced amino acid sequence of FIG.
 2. 22.Antibodies against the polypeptides of claim
 19. 23. An antagonistagainst the polypeptide of claim
 19. 24. A method for the treatment of apatient having need of ICE-LAP-3 comprising: administering to thepatient a therapeutically effective amount of the polypeptide of claim19.
 25. A method for the treatment of a patient having need of ICE-LAP-4comprising: administering to the patient a therapeutically effectiveamount of the polypeptide of claim
 19. 26. A method for the-treatment ofa patient having need to inhibit ICE-LAP-3 comprising: administering tothe patient a therapeutically effective amount of the antagonist ofclaim
 23. 27. A method for the treatment of a patient having need toinhibit ICE-LAP-4 comprising: administering to the patient atherapeutically effective amount of the antagonist of claim
 23. 28. Themethod of claim 24 wherein said therapeutically effective amount of thepolypeptide is administered by providing to the patient DNA encodingsaid polypeptide and expressing said polypeptide in vivo.
 29. The methodof claim 25 wherein said therapeutically effective amount of thepolypeptide is administered by providing to the patient DNA encodingsaid polypeptide and expressing said polypeptide in vivo.
 30. A methodfor detecting abnormal cell growth or the susceptibility to abnormalcell growth in a patient comprising: isolating nucleic acid sequencesencoding ICE-LAP-3 or 4 from a sample derived from a patient; anddetecting a mutation in the nucleic acid sequences encoding ICE-LAP-3 or4.